Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle ofthe virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-PheAsp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.The nucleocapsid protein N of vesicular stomatitis virus (VSV) tightly wraps the RNA genome and maintains the structural integrity and the template function of the negativestrand genome RNA (1). Within the virion this N-RNA template is associated with the RNA polymerase L and the transcription factor, phosphoprotein P, to form the transcribing ribonucleoprotein (RNP) complex. During transcription, the RNA polymerase complex (L and P) interacts with the N protein of the N-RNA template to transiently displace N and gain access to the genome RNA. During replication of the genome RNA, a soluble form of the N protein is required (2). This form of the N protein has been proposed to interact with nascently transcribed RNA chains from the RNP complex and to switch the mode of the RNA polymerase from transcription to replication (2). This results in the formation of an N-RNA complex containing full-length sense or antisense genome RNA. The precise mechanism by which the N protein recognizes the cognate sequence within the nascent RNA chain and continues to encapsidate during transcription remains unclear.Development of in vitro replication systems and studies on the structure and function of the N protein have provided some insight into the replication step of the virus' life-cycle (1). Although the N protein alone can initiate replication in vitro, albeit inefficiently, the presence of the P protein was found to greatly stimulate the reaction (3-5). Moreover, itThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact...

A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans ( Communicated by Lester 0. Krampitz, July 25, 1988 (received for review June 6, 1988 ABSTRACTThe mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.Evidence suggests that intermolecular (trans) splicing is used in a variety of organisms during the maturation of some mRNAs. This is particularly clear for trypanosomatid protozoans, where all mRNAs contain a common leader derived from a small nonpolyadenylylated miniexon transcript (for review, see ref. 1). A trans-splicing mechanism of leader addition is supported by the primary structure of the miniexon transcript and the existence of appropriate branched intermediates (2, 3). Recent observations indicate that transsplicing might also be used in the formation of mRNA for chloroplast ribosomal protein S12 (4) and in the maturation of certain actin mRNAs in Caenorhabditis elegans (5).In C. elegans, mRNAs derived from three of four actin genes contain a 22-nucleotide (nt) leader sequence that is not encoded within 15 kilobases (kb) of the actin genes. This leader sequence is found as the first 22 nt of an abundant 100-base RNA transcribed from within the 5S rRNA gene cluster (5). Several lines of evidence, including the demonstration of branched intermediates containing a portion of the 100-nt RNA, suggest that the 22-nt leader is acquired by trans-splicing (5, 16). In contrast to the situation in trypanosomes, only a subset of C. elegans mRNAs appear to contain the trans-spliced leader. Furthermore, because C. elegans actin genes contain multiple introns, trans-splicing apparently occurs in conjunction with conventional cis-splicing. As discussed by Krause and Hirsh (5) the use of trans-splicing in C. elegans raises the possibility that this mechanism could be widespread in eukaryotes and may be a regulatory mechanism in gene expression.We have recently described the isolation and characterization of cDNA and genomic clones encoding a 63-kDa protective antigen in the human parasitic nematode Brugia malayi, the causative agent of lymphatic filariasis (6, 7).Nuclease protection and primer-extension experime...

The phosphoprotein (P) of vesicular stomatitis virus (VSV) serotypes New Jersey [P(NJ)] and Indiana [P(I)] contains a highly conserved carboxy-terminal domain which is required for binding to the cognate N-RNA template as well as to form a soluble complex with the nucleocapsid protein N in vivo. We have shown that the deletion of 11 amino acids from the C terminal end of the P(I) protein abolishes both the template binding and the complex forming activity with the N protein. Within this region, there are conserved basic amino acid residues (R260 and K262) that are potential candidates for such interactions. We have generated mutant P proteins by substitution of these basic amino acid residues with alanine and studied their role in both transcription and replication. We have found that the R260A mutant failed to bind to the N-RNA template, whereas the K262A mutant bound efficiently as the wild-type protein. The R260A mutant, as expected, was unable to support mRNA synthesis in vitro in a transcription reconstitution reaction as well as transcription in vivo of a minigenome using a reverse genetic approach. However, the K262A mutant supported low level of transcription (12%) both in vitro and in vivo, suggesting that direct template binding of P protein through the C-terminal domain is necessary but not sufficient for optimal transcription. Using a two-hybrid system we have also shown that both R260A and K262A mutants interact inefficiently with the L protein, suggesting further that the two point mutants display differential phenotype with respect to binding to the template. In addition, both R260A and K262A mutants were shown to interact efficiently with the N protein in vivo, indicating that these mutants form N-P complexes which are presumably required for replication. This contention is further supported by the demonstration that these mutants support efficient replication of a DI RNA in vivo. Since the transcription defective P mutants can support efficient replication, we propose that the transcriptase and the replicase are composed of two distinct complexes containing (L-P2-3) and L-(N-P), respectively.

Specific in vivo interaction between the phosphoprotein (P) and the large polymerase protein (L) from the Indiana serotype of vesicular stomatitis virus was studied using a two-hybrid system. Transfection of CHO cells with plasmids encoding GALPIND and VPLIND fusion proteins resulted in an easily detectable level of CAT activity, indicating that PIND and LIND associate in vivo in the absence of other viral proteins. Mutational studies of PIND demonstrated that both domains I and II of PIND are important for PIND-LIND association. In addition, casein kinase II (CKII)-mediated phosphorylation within domain I of PIND was necessary for efficient association with LIND. We have also used the two-hybrid system to show PIND interaction with NIND in vivo. PIND and NIND associated more strongly than PIND and LIND. A similar strong association was observed in heterologous interaction studies between Indiana and New Jersey serotype P and N proteins. Mutational studies of PIND demonstrated that, unlike what was found for PNJ-NNJ association, only the C-terminal region of the P protein was important for efficient association with NIND. Like PNJ, CKII-mediated phosphorylation within domain I of PIND was not required for P-N association and, like NNJ, the C-terminal five amino acids of the NIND protein were critical for P association with N. These results demonstrate the importance of phosphorylation and specific domains of the P protein in its interaction with the L and N proteins, which are necessary for viral transcription and replication, respectively.

The phosphorylated state of the vesicular stomatitis virus phosphoprotein (P), an essential component of the virion-associated RNA polymerase complex, has been shown to be important for the transcriptional activity of the complex. Recent studies indicate that phosphorylation within the acidic domain of the P protein by cellular casein kinase II is necessary for its activity. In an attempt to identify the exact location of the cell kinase-mediated phosphorylation, we altered specific serine and threonine residues within the acidic domain of the New Jersey serotype of P protein by site-directed mutagenesis. The altered P proteins were then tested to determine what effect these mutations had on the phosphorylated state of the protein in vivo as well as its transcriptional activity in vitro. We report that serine residues 59 and 61 within the acidic domain of the P protein must be phosphorylated for it to be functionally active in a reconstituted transcription assay. These results demonstrate the importance of site-specific phosphorylation in the transcriptional activity of a negative-strand RNA viral phosphorprotein and the crucial role played by a cell protein kinase in this process.

DNA sequence analysis of genes encoding 5S rRNA in the human parasitic nematode Brugia malayi (B. malayi) indicates a surprising degree of heterogeneity. This variation in coding sequence is not accompanied by corresponding heterogeneity in flanking regions which are highly conserved. Six out of eight potential 5S coding regions differed; of these sequence variants, two were abundant in the B. malayi genome. Direct RNA sequence analysis indicated that one of these abundant variants accounts for most if not all of expressed 5S RNA at two stages of development.

The use of antisense RNA technology to inhibit virus gene expression has the potential to be a safe and effective antiviral therapy. In theory, antisense RNA would bind specifically to its target virus mRNA and block virus infection without interfering with the expression of endogenous genes or the normal functioning of the cell (Baltimore, 1988). The success of the antisense RNA approach depends on factors such as the vector and promoter chosen for stable integration and high level expression of the antisense RNA, the site of integration

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